1. Field of the Invention
The disclosure relates generally to the fields of probiotics and food.
2. Related Art
The activity and long term stability of many biological materials, such as proteins, enzymes and microbial cells may be affected by a number of environmental factors; for example, temperature, pH, the presence of water and oxygen or oxidizing or reducing agents. Generally, biological materials must be dried before or during mixing with other foodstuff ingredients. The drying process can often result in a significant loss in activity from mechanical, chemical, and osmotic stresses induced by the drying process. Loss of activity occurs at many distinct stages, including drying during initial manufacturing, feed preparation (high temperature and high pressure), transportation and long term storage (temperature and humid exposure), and after consumption and passage in the gastrointestinal (GI) track (exposure to low pH, proteolytic enzymes and bile salts). Manufacturing food or feedstuffs with live cell organisms or probiotics is in particular challenging, because the probiotics are very sensitive to the drying process and to temperature and moisture conditions of the foodstuff. Another concern is the probiotic resistance in the acid environment in the stomach and its successful colonization of the intestine.
Probiotic microorganisms (probiotics) are living microorganisms, which upon ingestion in certain numbers, exert health benefits beyond basic nutrition. The beneficial effects that probiotics may induce are numerous. Few examples are; the reduction of lactose intolerance, the inhibition of pathogenic bacteria and parasites, the reduction of diarrhea, activity against Helicobacter pylori, the prevention of colon cancer, the improvement or prevention of constipation, the in situ production of vitamins, the modulation of blood lipids, and the modulation of host immune functions. In domesticated and aquatic animals they also can improve growth, survival and stress resistance associated with diseases and unfavorable culture conditions. Therefore, there is considerable interest in including probiotics into human foodstuffs and into animal feed.
Many probiotics exhibit their beneficial effect mainly when they are alive. Hence, they need to survive the manufacturing process and shelf life of the food, and upon consumption of the food where they need to pass through the gastro-intestinal tract before reaching their place of colonization. Although many commercial probiotic products are available for animal and human consumptions, most of them lost their viability during the manufacture process, transport, storage and in the animal GI tract (see the viability studies of several probiotic products by (Hughes and Hillier 1990; Shah 2000). To compensate for such loss, an excessive quantity of probiotics is included in the product in anticipation that a portion will survive and reach their target. In addition to questionable shelf-life viability for these products, such practices are certainly not cost-effective. Alternatively, the probiotic microorganisms can be encapsulated in protective microenvironments. Generally, current microencapsulation and enteric coating techniques involve applying a film forming substance, usually by spraying liquids containing sugars or proteins onto the dry probiotics (Ko and Ping WO 02/058735). However, coating the microencapsulated probiotics with moisture protecting layers is an expensive process, and generally several layers must be added, to avoid water entering the microcapsules. In addition, it is extremely difficult to remove the added liquid in the coating substance without a corresponding decrease in shelf life.
Various protective agents have been used in the art, with varying degrees of success. These include proteins, certain polymers, skim milk, glycerol, polysaccharides, oligosaccharides and disaccharides. Disaccharides, such as sucrose and trehalose, are particularly attractive cryoprotectants because they are actually help plants and microbial cells to remain in a state of suspended animation during periods of drought. Trehalose has been shown to be an effective protectant for a variety of biological materials, both in ambient air-drying and freeze-drying (Crowe et al. 1998). However, there are some drawbacks associated with the use of sugars as the sole cryoprotectant. For example, large amounts of sugars (often greater than 60% by weight) must be used to preserve the biological materials during the drying process. This is costly. More serious problems associated with the use of sugars include their readiness to form crystals when the material is dried below its freezing point, and the low glass transition temperature which causes instability of the preserved biological materials at high temperatures, and/or in humid environments. Further, high concentration of sugars reduces the solubility of other solutes in the system and at the same time renders the system extremely difficult to dry.
Accordingly, it has been proposed to dry sugar-based probiotic systems by foam formation in a very thin layer (Bronshtein WO2005117962), or to use combinations of sugars with a polymeric gelling agent, such as alginate, chitosan, carboxymethylcellulose or carboxyethylcellulose. Cavadini et al. (EP 0 862 863) provide a cereal product comprising a gelatinized starch matrix including a coating or a filling. The probiotic is included with the coating. According to that process, spray-dried probiotics are mixed with a carrier substrate, which may be water, fat or a protein digest. The mixture is then sprayed onto the cereal product and the whole product is dried again. Re-hydrating of the already dried bacteria and the additional coating/drying process is costly and damaging to the bacteria.
Kenneth and Liegh (U.S. Pat. No. 6,900,173) describe the manufacturing of multivitamin protein and probiotic bar for promoting an anabolic state in a person. The dried probiotic bacteria are blended in sugar syrup and several other constituents, and the resultant mixture is then extruded and cut into bars. However, the document does not disclose any process or composition that will improve viability or long-term stability of probiotics in the nutritional bars and there is no indication that the bacteria even survive the process.
Ubbink et al. (US 2005/0153018) disclose the preservation of lactic acid bacteria in moist food. The spray-dried bacteria are added to a composition comprising fats, fermented milk powder and saccharides. That composition is then used as the filling of a confectionary product. The subject matter described in that document avoids the detrimental effects of water by embedding the probiotics in fat or oil rich matrix. However, fat based coating and preserving materials do not withstand long term exposure to humid conditions.
Giffard and Kendall (US 2005/0079244) disclose a foodstuff in the form of a dried or semi-moist ready-to-eat kibble or powder mix, which contains a combination of a probiotic, prebiotic and a coating of colostrum. Prior to mixing in the food stuff, the probiotic is coated or encapsulated in a polysaccharide, fat, starch, protein or in a sugar matrix using standard encapsulation techniques. Similar to the above disclosure, the negative effects of water were avoided by embedding the probiotics in a matrix rich in fat or oil.
Farber and Farber (WO 03/088755) describe an oral delivery system for functional ingredients uniformly dispersed in a matrix. The matrix components include a sugar, a carbohydrate, a hydrocolloid a polyhydric alcohol and a source of mono- or divalent cations. The delivery system is extruded or molded into a final shape with a moisture content of between 15% and 30% by weight. This type of matrix provides very little protection to the probiotics mostly under refrigerated conditions. No description or direction was provided as to how probiotic bacteria are stabilized during manufacturing or for prolonged storage at room temperatures.
Porubcan (US 2004/0175389) discloses a formulation for protecting probiotic bacteria during passage through the stomach, whilst permitting their release in the intestine. The formulation has also a low water activity and correspondingly long shelf life. The capsule includes a water-free mixture of probiotic bacteria with monovalent alginate salts, and an enteric coating (e.g., gelatin or cellulose encapsulation). Upon contact with acidic environment, the outer shell of the capsule turned into a gel, which provides a protecting barrier against proton influx into the capsule core. However, this composition is only useful for large particles such as tablets and capsules subjected to storage conditions of very low water activity and further require storage in nitrogen-flushed or vacuum-sealed containers. McGrath and Mchale (EP 1382241) describe a method of delivering a microorganism to an animal. The micro-organism is suspended in a matrix of cross-linked alginate and cryopreservant (trehalose or lactose, or a combination of both). The matrix is then freeze or vacuum dried to form dry beads containing live probiotics with a shelf-life stability up to 6 months but only under refrigerated conditions. Here again, no description or direction was provided as to how probiotic bacteria are stabilized during manufacturing or for prolonged storage at room temperatures and high humidity conditions.
None of the above compositions provide a mixture that can effectively protect the probiotic in both drying processes and long-term storage at elevated temperatures and varying degrees of humidity. Therefore, there is an urgent need for such a composition that can effectively protect the probiotic bacteria during manufacturing, long-term storage at elevated temperatures and humidity and during gastrointestinal passage. There is a need also for a drying process that is cost-effective and capable of entrapping and stabilizing probiotics in the protective mixture with minimal viability loss at the end of the entire operation. There is a need for a protective mixture that provides protection in the animal stomach while allowing the release of the probiotic along the intestinal tract. There is also a need for a protective mixture that contains only approved ingredients generally regarded as safe (GRAS), and is less costly than those presently being used.
The subject matter described herein overcomes these needs and provides a composition and process for producing a composition that provides probiotic bacteria that are stable for long periods of time even at elevated temperatures and varying degrees of humidity.
It is, in particular, a purpose of the present disclosure to describe viable probiotic cultures that are substantially stable at room temperature and high humidity conditions thereby obviate the need for refrigeration or storage under vacuum or oxygen free environment.